The Angiotensin II Type 2 Receptor AT2R is a member of the anti-inflammatory/vasodilative branch of the renin-angiotensin system (RAS). AT2R-activation ameliorates cardiovascular diseases and stroke, attenuates cancers and exerts a neuroprotective role (1-17).
AT2R is a transmembrane receptor protein comprising a sequence of 363 amino acids which form seven-transmembrane domains. The three-dimensional structure of AT2R has not yet been resolved, but it contains five potential glycosylation sites and a conserved lysine residue (Lys 199 or K199) that is critical for ligand-protein interaction. AT2R also contains a potential protein kinase C phosphorylation site in the second intracellular loop (18).
AT2R belongs to the G-Protein Coupled Receptor (GPCR) family of proteins. AT2R activation stimulates various protein phosphatases (e.g., SHP1, MKP1 and PP2A) and inhibits cancer cell growth. AT2R-mediated activation of the bradykinin/nitric oxide/cGMP pathway and the prostaglandin I2-IP receptor pathway contribute to its vasodilatory effects (19-23). However, many of the signaling mechanisms activated by the AT2R are G-protein independent and involve direct interactions between AT2R and other cellular proteins. AT2R interacts with a family of AT2R-interacting proteins (ATIPs) involved in neuronal differentiation, vascular remodeling and tumor suppression via its C-terminal cytoplasmic domain (CCD) (24). It has been shown that the AT2R interacts with the ErbB family receptors and Na+/H+ exchanger NHE6 via its third intracellular loop (ICL) and the CCD, and that the third ICL of the AT2R is involved in attenuating Angiotensin II Type 1 Receptor (AT1R) signaling (25, 26). AT2R-mediated apoptosis also requires the third ICL (27, 28). Interestingly, deletion of the CCD reduces affinity of AT2R to Angiotensin II (Ang II), but increases its affinity to the peptide ligand CGP42112A and enhances Ang II-induced cGMP reduction (29, 30). These observations highlight the roles of the third ICL and the CCD in AT2R signaling.
AT2R down-regulation is seen in Parkinson's Disease (PD) (31). Although not much is known about the role of AT2R in PD, it is known that AT2R activation causes differentiation of dopaminergic neurons from mesencephalic precursors (32). Additionally, AT2R activation is neuroprotective to cultured mid-brain dopaminergic neurons, whereas use of an AT2R antagonist eliminates the neuroprotective effects (33).
Early studies from Mendelsohn et al. (1988) and Unger et al. (1988) established, using biochemical and pharmacological approaches, the existence of a renin-angiotensin system in the brain (34, 35). The various components (e.g., angiotensin-converting enzyme (ACE), Ang II and Ang II receptors) are found in areas of the brain involved in the regulation of fluid and electrolyte balance and in the regulation of arterial pressure (36, 37), as well as in structures involved in cognition, behavior and locomotion. Interestingly, all of these components, and in particular AT2R, are highly expressed during fetal life. This suggests that they could play important roles during development. As reported by Nuyt et al. based on studies conducted in fetal and neonatal rats, AT2R mRNA appeared early (e.g., as early as embryonic day 13) in the differentiating lateral hypothalamic area, but transiently in various developing/differentiating brain structures (38). In most areas, the ontogeny of AT2R mRNA expression is highly correlated with the maturation and differentiation of the different areas themselves (as in the cerebellum, inferior olivary complex, and medullary motor nuclei innervating the tongue, perioral, and jaw muscles, where AT2R expression dramatically diminished in the mature neurons).
From studies conducted in cell lines, it appears that activation of AT2R during development is involved in neurite elongation, neuron migration, neuronal death and survival balance, as well as in the establishment and maintenance of synaptic connections. In the adult rat, AT2R was found at high levels in the medulla oblongata (which controls autonomous functions), in septum and amygdala (which are associated with anxiety-like behavior), in the thalamus (which is associated with sensory perception), in the superior colliculus (which controls eye movements in response to visual information and is linked to blink hyperexcitability in Parkinson's), as well as in the subthalamic nucleus and in the cerebellum (areas associated with learning of motor functions) (39, 40, 41).
According to Bottari et al., AT2Rs are found on neurons, but not on astrocytes or glial cells. The presence of AT2R in restricted brain areas of the adult and its wide distribution in the fetus (in many differentiating structures and nuclei) are indicative of a role in neuronal function and neuronal development respectively (42). Accordingly, using cells of neuronal origin and models of neuronal regeneration, AT2R was found to be involved in the regulation of apoptosis and cell differentiation. Apart from its transient expression in many structures during development, expression of AT2R increases in the brain after cellular damage, which shows that it plays a role in neuronal wound healing. In addition to neuronal differentiation, which is of paramount importance in nerve regeneration, AT2R also stimulates differentiation of hematopoietic cells, a key process during regeneration and reconstruction (31).
Ischemic damage is characterized by infiltration of a number of hematopoietic cells such as platelets, macrophages, and leukocytes (43). Significantly, AT2R has the capacity to induce differentiation of human monocytes into dendritic cells (44), indicating a potential protective effect. Confirming this protective effect of AT2R is the observation that ischemic damage was found to be greater in mice with hematopoietic cells deleted in AT2R expression (45). These findings show that expression and activation of AT2R in hematopoietic cells is part of its beneficial effect following brain injury (46).
Renal dopamine D1-like receptors (D1Rs) and AT2Rs are important natriuretic receptors counter-balancing AT1R-mediated tubular sodium reabsorption. In uninephrectomized, sodium-loaded Sprague-Dawley rats, direct renal interstitial infusion of the highly selective D1R agonist fenoldopam induced a natriuretic response that was abolished by the AT2R-specific antagonist PD-123319 or by the microtubule polymerization inhibitor nocodazole but not by the actin polymerization inhibitor cytochalasin D. The results demonstrate that D1R-induced natriuresis requires AT2R recruitment to the apical plasma membranes of renal proximal tubule cells in a microtubule-dependent manner involving an adenylyl cyclase/cAMP signaling pathway. These studies provide novel insights regarding the mechanisms whereby renal D1Rs and AT2Rs act in concert to promote sodium excretion in vivo (47).
Treatments of primary neurons with Compound 21 (C21), an AT2R agonist, improved functional recovery in experimental spinal cord injury through promotion of axonal plasticity and through neuroprotective and anti-apoptotic mechanisms (48).
Even though AT2R belongs to the GPCR family of proteins, its signaling mechanisms are atypical and remain elusive. Activated AT2R induces a vasodilator cascade of bradykinin (BK)/Nitric Oxide/cGMP, stimulates various protein phosphatases (e.g., SHP1, MKP1 and PP2A) and inhibits cancer cell growth (19-23). AT2R also interacts with a family of AT2 receptor interacting proteins (ATIPs) involved in neuronal differentiation, vascular remodeling and tumor suppression via its CCD (49, 50). Chronic AT1 Receptor blocker (ARB) treatment can result in redirecting Ang II to AT2R that is usually co-expressed with AT1R in cardiovascular tissues, leading to increased AT2R activation, and enhanced AT1R-AT2R cross-talk. In AT2R knock-out mice, ARBs failed to attenuate acute-phase post-infarction remodeling indicating that AT2R is required for the cardioprotective effects of ARBs (17).
Cardiovascular protective effects of AT2R are highlighted by the fact that moderate cardiac-specific AT2R overexpression protects the heart from ischemic injury (16).
The inflammatory cascade contributing to the development of cardiovascular disease (CVD) has been rapidly elucidated over the past decade, inspired by the marked increase in disease prevalence. To put this in perspective, nearly 70% of all Type 1 Diabetes Mellitus (T1DM) fatalities are attributed to the condition (51). Increased activation of the pro-inflammatory AT1R is seen in cardiovascular disease and hypertension (3). In general, increased AT1R activation up-regulates pro-inflammatory and pro-cancerous proteins such as nf-kb, IL-6 (52, 53, 54),
Diabetic nephropathy is marked by increased basal levels of certain cytokines (e.g., TNF-alpha, IL-6) and therefore experimental treatments have focused on modulating these same markers. Multiple studies have revealed that levels of cytokines in serum and urine are positively correlated with the progression of the disease. Particularly related to the pathogenesis of nephropathy, molecules such as IL-6 have been identified as being responsible for altering the permeability of vascular endothelial cells and the development of basement membrane thickening, respectively (55).
Chronic activation of RAS systemically and locally elevates Ang II. Ang II then binds to AT1R and induces signaling pathways that promote muscle constriction, salt and water retention, fibrosis, hypertrophy and hyperplasia that underlie many of the metabolic diseases and poor cardiovascular and renal prognosis. Blockade of RAS can be exerted at multiple levels: via inhibition of Renin, ACE, or AT1R signaling (1, 2, 5, 9). Efficient RAS blockers at all these levels have been developed and are currently in use to block over-activation of RAS and to offer protection from RAS-related metabolic diseases including diabetes (2).
However, evidence from randomized clinical trials such as the Aliskiren Trial in Type 2 Diabetes Using Cardio-Renal Endpoints (ALTITUDE) and the Ongoing Telmisartan Alone and in Combination With Ramipril Global Endpoint Trial (ONTARGET) shows that dual RAS blockade was not beneficial compared to monotherapy in preventing serious outcomes in patients with known vascular disease or diabetes with end-organ damage (56-58). Clinical evidence supporting the association of RAS inactivation to renal diseases and basic research on RAS have begun to unveil the intricate self-regulatory signaling loops that fine-tune RAS activation and the adaptive/protective role of RAS in many tissues (9). In this context, Ang II manifests its vasodilative/cardiovascular protective/anti-inflammatory effects when it activates AT2R.
Mitochondria also express a local angiotensin system (MAS). Importantly, AT2R located in the inner membrane of mitochondria plays a significant role in mediating mitochondrial respiration. It is known that during the aging process, mitochondrial AT2R expression is reduced, while the expression of the pro-inflammatory AT1R is increased (62). The critical role of MAS in aging indicates that this system plays a role in Alzheimer's Disease (AD) development. In further support, it has been shown that amyloid-beta leads to the increased oligomerization and loss of function of the AT2R receptor, which is thought to contribute to pathogenesis of the disease (63, 64).
AT1R blockers (ARBs) have been reported to reduce age related mitochondrial dysfunction, attenuate hypertension-induced renal mitochondrial dysfunction, and protect against cardiac mitochondrial dysfunction in the setting of acute ischemia (62, 65). Inhibition of AT1R by ARBs theoretically allows more Ang II to bind and activate AT2R. Therefore, elevation of the opposing AT2R system will provide additional improvements in mitochondrial function. Disruption of AT1R was associated with an increased number of mitochondria and up-regulation of the prosurvival genes nicotinamide phosphoribosyltransferase (Nampt) and sirtuin 3 (Sirt3) in the kidney, leading to marked prolongation of life span in mice (66). Of these genes, Sirt3 is known to regulate AD-mediated stress (67).
NHE6 is a mitochondrial protein located in the inner membrane of mitochondria known to improve cognition and memory, and mutations in the NHE6 gene are linked with various neurological disorders such as autism and Christianson's Syndrome (68-70). It has been shown that AT2R interacts with NHE6 via its third ICL. AT2R inhibits AT1R-mediated threonine/tyrosine phosphorylation of NHE6 (71-73). This indicates that AT1R-mediated phosphorylation is a tag for degradation that is prevented via the AT2R-NHE6 interaction.
MCL-1 (myeloid leukemia cell differentiation protein) is a protein that is a member of the Bcl-2 family (74). There are two distinct variants of MCL-1, based on alternative splicing: a long form and two shorter isoforms. The long form (MCL-1L) contains 312 residues, while the short isoforms (MCL-1S) is 271 residues, with the 41 residue difference occurring at the C terminus. MCL-1L contains the standard domains found in the Bcl-2 family including BH1, BH2, BH3, and a transmembrane domain. In contrast, MCL-1S only contains the BH3 domain. This alternative splicing leads to two vastly different biological functions for MCL-1L and MCL-1S. Specifically, MCL-1L is known to be anti-apoptotic while the MCL-1S in complete contrast is pro-apoptotic (75, 76). The BH3-like domain region of MCL-1S can bind and dimerize with MCL-1L (77). This interaction inhibits MCL-1 biological activity and therefore MCL-1S is an antagonist to MCL-1L.